Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes

ABSTRACT

The present invention relates to an in vitro diagnostic method for malaria in an individual comprising placing a tissue or a biological fluid taken from an individual in contact with a molecule or polypeptide composition, wherein said molecule or polypeptide composition comprises one or more peptide sequences bearing all or part of one or more T epitopes of the proteins resulting from the infectious activity of  P. falciparum , under conditions allowing an in vitro immunological reaction to occur between said composition and the antibodies that may be present in the tissue or biological fluid, and in vitro detection of the antigen-antibody complexes formed. The invention further relates to a polypeptide comprising at least one T epitope from a liver-stage specific protein produced by  P. falciparum  and a vaccine composition directed against malaria comprising a molecule having one or more peptide sequences bearing all or part of one or more T epitopes resulting from the infectious activity of  P. falciparum  in the hepatic cells.

This application is a divisional of application Ser. No. 08/462,625filed on Jun. 5, 1995 now U.S. Pat. No. 6,319,502, which is a divisionalof application Ser. No. 08/098,327 filed on Nov. 24, 1993 now U.S. Pat.No. 6,270,771, which is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/FR92/00104 filed on Feb. 5, 1992,which International Application was not published by the InternationalBureau in English.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The parasites responsible for malaria in man, including in particularPlasmodium falciparum and Plasmodium vivax to mention only the principalones among them, exhibit different morphologies in the human host andexpress different antigens as a function of their localization in theorganism of the infected host. The morphological and antigenicdifferences shown by these parasites during their life cycle in manmakes it possible to define at least four distinct stages ofdevelopment.

(2) Description of the Prior Art

The very first stage of development of the parasite in man correspondsto the sporozoite form introduced into the blood of the host by bites ofinsect carriers of the parasite. The second stage corresponds to thepassage of the parasite into the liver and to the infection of thehepatic cells in which the parasites develop to form the hepaticschizonts which burst to release the hepatic merozoites. The third stageis characterized by the infection of the blood erythrocytes by theasexual forms (merozoites) of the parasite; this erythrocytic form ofdevelopment of the parasite corresponds to the pathogenic phase of thedisease. The fourth stage corresponds to the formation of the sexualforms (or gametocytes) which will become the extracellular gametes inthe mosquito.

It is known that very many studies have been undertaken in order toisolate from the strains of parasites infective for a human hostpolypeptide fractions to provide for the needs of the in vitro diagnosisof malaria by detection of the corresponding antibodies, on the onehand, and to attempt to vaccinate against malaria, on the other.

For example, libraries of cloned cDNAs derived from the sporozoites ofPlasmodium falciparum have been established by ENEA et al. (1984) ,Science, vol. 225, 628–630. It was recognized that these librariesincluded clones capable of expressing immunogenic polypeptidescontaining repetitive units of 4 amino acids specific for thecircumsporozoite antigen (of P. falciparum).

However, little work has been done on the hepatic forms of the parasitesresponsible for malaria. The morphology of the hepatic forms wasdescribed for the first time in 1948 on biopsies taken from infectedhuman volunteers (Trans. Roy. Soc. Trop. Med. Hyg., 41, 785 (1948). Itwas possible to describe an antigen specific for the hepatic stage of P.falciparum in the liver of South American monkeys insensitive to theblood forms of the parasite, but in which the hepatic forms can develop(Am. J. Trop. Med. Hyg., 33 (3) 336–341 (1984).

The detection of the localization of the liver-specific antigens(designated hereafter by LS antigens for “Liver Stage” antigens) wascarried out by immunofluorescence throughout the maturation steps of theschizont. They are localized at the periphery of the parasite 5 to 40microns in size; subsequently they are distributed between the cytomersor packets of merozoites, when the schizonts attain between 50 and 100microns. They are different from the surface antigens of the sporozoitesand the antigens shared by the schizonts of the blood and the liverwhich give an internal immunofluorescence image of the parasite.

Although it is now possible to culture hepatic forms of P. falciparum inhuman hepatocytes (Science, 227, 440 (1985)), the low numbers of matureforms of the parasite obtained by the in vitro and in vivo culturemethods do not allow the biochemical analysis of the antigen produced atthe hepatic stage.

It has also been observed that the individuals suffering from malariapossess a very high level of antibodies directed against the LSA. The LSantigens seem to be very potent immunogens, among the most potent of allof the antigens synthesized at the various stages of development of theparasite.

BRIEF SUMMARY OF THE INVENTION

One of the objectives of the present invention is precisely to providenovel compositions for the vaccination of humans against the malariacaused by P. falciparum.

The objective of the invention is also the in vitro diagnosis of theinfection of an individual by P. falciparum under more sensitiveconditions than present methods allow.

A molecule expressed specifically during the hepatic phase has beenidentified by screening a library of genomic DNA cloned in an expressionvector with polyclonal sera (GUERIN-MARCHAND, C. et al.; Nature, 329,164–167 (1987)). This molecule represents a part of an antigen calledLSA (Liver Stage Specific antigen), and is constituted of repetitivemotifs of 17 amino acids and seems to be very immunogenic under thenatural conditions of exposure to the disease.

These repetitive motifs (SEQ ID NO: 1) of 17 amino acids are representedby the formula:

Leu-Ala-Lys-Glu-Lys-Leu-Gln-X-Gln-Gln-Ser-Asp-Leu-Glu-Gln-Glu-Arg inwhich X is Glu or Gly.

The subject of the invention is peptide sequences specific for thehepatic stages of P. falciparum which bear epitopes capable ofstimulating the T lymphocytes (in particular the cytotoxic lymphocytes).

The invention relates more particularly to molecules or to peptide orpolypeptide compositions characterized by the presence in theirstructure of one or more peptide sequences bearing all or part of one ormore T epitope(s) (epitopes implicated in the stimulation of the Tlymphocytes) and, optionally, other epitopes, in particular B epitopes(epitopes corresponding to the antibodies produced by B lymphocytes),characteristic of the proteins resulting from the infectious activity ofP. falciparum in the liver cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference will be made in what follows to the Figures in which:

FIG. 1 (SEQ ID NO: 31) presents a recombinant protein of the inventionof 316 amino acids, designated hereafter as antigen 536 or proteinLSA-R-NR,

FIG. 2 (SEQ ID NO: 32) provides the nucleotide sequence of one of therecombinant nucleic acids studied (clone DG536) and which codes for thepolypeptide LSA-R-NR,

FIG. 3 (SEQ ID NO: 24) presents a polypeptide of the invention of 151amino acids, designated hereafter as antigen 729S,

FIG. 4 (SEQ ID NO: 33) corresponds to the nucleotide sequence of theclone DG729S which codes for the polypeptide of FIG. 3 (EcoRI linkers inbold type),

FIG. 5 presents the polypeptide sequences (SEQ ID NOS: 23 and 26–28) ofthe antigens LSA-Ter, 729S-NRI, 729S-NRII, 729S-Rep,

FIG. 6 (SEQ ID NO: 34) presents the 5′ end of the nucleotide sequence ofthe LSA gene,

FIGS. 7A–7C (SEQ ID NOS: 35–37) presents the coding sequence of the 5′end of the LSA gene and the corresponding polypeptide sequence,

FIG. 8 (SEQ ID NO: 38) describes the 3′ end of the LSA gene,

FIGS. 9A–9D (SEQ ID NOS: 39–42) gives the sequence of the 3′ end of theLSA gene as well as the corresponding polypeptide sequence,

FIGS. 10A–10D (SEQ ID NOS: 43–46) repeats the sequences given in FIGS.9A–9D, up to the termination codon stop and the termal amino acid.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention relates to any molecule or polypeptidecomposition bearing at least one peptide sequence bearing all or part ofone or more epitopes characteristic of a protein produced in thehepatocytes infected by P. falciparum, and more particularly bearing allor part of one or more T epitope(s) of the proteins produced at thehepatic stage of P. falciparum, characterized in that this peptidesequence is represented by all or part of the amino acid sequence shownin FIGS. 9A–9D or FIGS. 10A–10D, and corresponds to the 3′ end of theLSA gene.

More particularly, the subject of the invention is any molecule orpolypeptide composition bearing at least one peptide sequence bearingall or part of one or more epitopes characteristic of a protein producedin the hepatocytes infected by P. falciparum, and more particularlybearing all or part of one or more T epitope(s) of the proteins producedat the hepatic stage of P. falciparum, characterized in that thispeptide sequence is represented by all or part of the sequence of thelast 279 amino acids shown in FIGS. 10A–10D, this amino acid sequencebeing optionally preceded by all or part of one or more of the sequencesof 17 amino acids (SEQ ID NOS: 2–18) of formula:

X₁DLEQX₂RX₃AKEKLQX₄QQ QX₁DLEQX₂RX₃AKEKLQX₄Q QQX₁DLEQX₂RX₃AKEKLQX₄X₄QQX₁DLEQX₂RX₃AKEKLQ QX₄QQX₁DLEQX₂RX₃AKEKL LQX₄QQX₁DLEQX₂RX₃AKEKKLQX₄QQX₁DLEQX₂RX₃AKE EKLQX₄QQX₁DLEQX₂RX₃AK KEKLQX₄QQX₁DLEQX₂RX₃AAKEKLQX₄QQX₁DLEQX₂RX₃ X₃AKEKLQX₄QQX₁DLEQX₂R RX₃AKEKLQX₄QQX₁DLEQX₂X₂RX₃AKEKLQX₄QQX₁DLEQ QX₂RX₃AKEKLQX₄QQX₁DLE EQX₂RX₃AKEKLQX₄QQX₁DLLEQX₂RX₃AKEKLQX₄QQX₁D DLEQX₂RX₃AKEKLQX₄QQX₁in which:

-   -   X₁ is “Ser” or “Arg”,    -   X₂ is “Glu” or “Asp”    -   X₃ is “Arg” or “Leu”    -   X₄ is “Glu” or “Gly”

Consequently, the invention relates more particularly to any molecule orpolypeptide composition bearing at least one peptide sequence bearingall or part of one or more epitopes characteristic of a protein producedin the hepatocytes infected by P. falciparum, and more particularlybearing all or part of one or more T epitope(s) of the proteins producedat the hepatic stage of P. falciparum, characterized in that thispeptide sequence is represented by all or part of the following aminoacid sequence (SEQ ID NO: 19):

RKADTKKNLERKKEHGDILAEDLYGRLEIPAIELPSENERGYYIPHQSSLPQDNRGNSRDSKEISIIEKTNR ESITTNVEGRRDIHKGHLEEKDGSIKPEQKEDKSthis amino acid sequence being optionally preceded by all or part of oneor more sequences of 17 amino acids (SEQ ID NOS: 2–18) of formula:

X₁DLEQX₂RX₃AKEKLQX₄QQ QX₁DLEQX₂RX₃AKEKLQX₄Q QQX₁DLEQX₂RX₃AKEKLQX₄X₄QQX₁DLEQX₂RX₃AKEKLQ QX₄QQX₁DLEQX₂RX₃AKEKL LQX₄QQX₁DLEQX₂RX₃AKEKKLQX₄QQX₁DLEQX₂RX₃AKE EKLQX₄QQX₁DLEQX₂RX₃AK KEKLQX₄QQX₁DLEQX₂RX₃AAKEKLQX₄QQX₁DLEQX₂RX₃ X₃AKEKLQX₄QQX₁DLEQX₂R RX₃AKEKLQX₄QQX₁DLEQX₂X₂RX₃AKEKLQX₄QQX₁DLEQ QX₂RX₃AKEKLQX₄QQX₁DLE EQX₂RX₃AKEKLQX₄QQX₁DLLEQX₂RX₃AKEKLQX₄QQX₁D DLEQX₂RX₃AKEKLQX₄QQX₁in which:

-   -   X₁ is “Ser” or “Arg”,    -   X₂ is “Glu” or “Asp”    -   X₃ is “Arg” or “Leu”    -   X₄ is “Glu” or “Gly”

Thus the present invention relates in particular to the peptide sequenceshown in FIG. 1. This sequence is constituted of 316 amino acids. At the5′ end 209 amino acids are found organized in repeats of 17 amino acidscorresponding to the formulae indicated above. At the 3′ end, repeatstotalling 107 amino acids are found.

The invention relates more particularly to any polypeptide characterizedby all or part of the following amino acid sequence (SEQ ID NO: 20):

LQEQQRDLEQRKADTKKNLERKKEHGDILAEDLYGRLEIPAIELPSENER GYYIPHQSSLPQDNRGNSRDSKEISIIEKTNRESITTNVEGRRDIHKGHLEEK KDG SIKPEQKEDKS

A preferred polypeptide of the invention is represented by all or partof the following amino acid sequence (SEQ ID NO: 21):DTKKNLERKKEHGDILAEDLYGRLEIP(this polypeptide being designated hereafter by the expression LSA-NR(LSA-non-repeated), or also by any sequence derived from the precedingsequence and modified by the substitution of maximally 40% of the aminoacids while retaining its physiological activity such as the inductionof a response of the T lyphocytes, in particular the cytotoxic Tlymphocytes.

Another particularly preferred polypeptide of the invention ischaracterized by all or part of the following amino acid sequence (SEQID NO: 22):ERRAKEKLQEQQRDLEQRKADTKK(this polypeptide being designated hereafter by the expression LSA-J, orLSA-junction, since it overlaps the repetitive part and thenon-repetitive part of the molecule shown in FIG. 1).

Another preferred peptide, designated LSA-TER, is the following (SEQ IDNO: 23):NSRDSKEISIIEKTNRESITTNVEGRRDIHK

These last three polypeptides are more particularly useful on account ofthe amphipaticity which characterizes them, and because of theirthree-dimensional conformation according to the predictions made by theprocedure of Chou and Fassmann.

The subject of the invention is also any molecule or polypeptidecomposition bearing at least one peptide sequence bearing all or part ofone or more epitope(s) characteristic of a protein produced at thesporozoite, hepatic and blood (erythrocytic) stages of P. falciparum,and more particularly bearing one or more T epitopes, characterized inthat this peptide sequence is represented by all or part of thefollowing amino acid sequence (SEQ ID NO: 24):

RDELFNELLNSVDVNGEVKENILEESQVNDDIFNSLVKSVQQEQQHNVEE KVEESVEENDEESVEENVEENVEENDDGSVASSVEESIASSVDESIDSSIEEN VAPTVEEIVAPTVEEIVAPSVVEKCAPSVEESVAPSVEESVAEMLKERshown in FIG. 3 and designated hereafter as the polypeptide 729S.

More particularly, the subject of the invention is the amino acidsequence derived from the preceding sequence and characterized by all orpart of the following amino acid sequence (SEQ ID NO: 25):RDELFNELLNSVDVNGEVKENILEESQVNDDIFNSLVKSVQQEQQHN

According to another advantageous embodiment of the invention, sequencesof interest derived from the amino acid sequence of the polypeptide 729Sare the following (SEQ ID NOS: 26–28):

DELFNELLNSVDVNGEVKENILEESQ, LEESQVNDDIFSNSLVKSVQQEQQHNV,VEKCAPSVEESVAPSVEESVAEMLKER.

These sequences are designated 729S-NRI, 729S-NRII, 729S-Rep,respectively.

The subject of the invention is also any molecule or polypeptidecomposition comprising at least one peptide sequence bearing all or partof one or more epitopes characteristic of a protein produced in thehepatocytes infected by P. falciparum, characterized in that thispeptide sequence is represented by all or part of the amino acidsequence shown in FIGS. 7A–7C

Consequently, the subject of the invention is more particularly anymolecule or polypeptide composition comprising at least one peptidesequence bearing all or part of one or more epitopes characteristic of aprotein produced in the hepatocytes infected by P. falciparum, andbearing more particularly all or part of one or more T epitope(s) of theproteins produced at the hepatic stage of P. falciparum, characterizedin that this peptide sequence is represented by all or part of thesequence of the first 153 amino acids shown in FIGS. 7A–7C, this aminoacid sequence being optionally followed by all or part of one or moresequences of 17 amino acids (SEQ ID NOS: 2–18) of formula:

X₁DLEQX₂RX₃AKEKLQX₄QQ QX₁DLEQX₂RX₃AXEKLQX₄Q QQX₁DLEQX₂EX₃AKEKLQX₄X₄QQX₁DLEQX₂RX₃AKEKLQ QX₄QQX₁DLEQX₂RX₃AKEKL LQX₄QQX₁DLEQX₂RX₃AXEKKLQX₄QQX₁DLEQX₂RX₃AKE EKLQX₄QQX₁DLEQX₂RX₃AK KEKLQX₄QQX₁DLEQX₂RX₃AAKEKLQX₄QQX₁DLEQX₂RX₃ X₃AKEKLQX₄QQX₁DLEQX₂R RX₃AKEKLQX₄QQX₁DLEQX₂X₂RX₃AKEKLQX₄QQX₁DLEQ QX₂RX₃AKEKLQX₄QQX₁DLE EQX₂RX₃AKEKLQX₄QQX₁DLLEQX₂RX₃AKEKLQX₄QQX₁D DLEQX₂RX₃AXEKLQX₄QQX₁in which:

-   -   X₁ is “Ser” or “Arg”,    -   X₂ is “Glu” or “Asp”    -   X₃ is “Arg” or “Leu”    -   X₄ is “Glu” or “Gly”

The invention also relates to any molecule or polypeptide compositioncomprising at least one peptide sequence bearing all or part of one ormore epitopes characteristic of a protein produced in the hepatocytesinfected by P. falciparum, and bearing more particularly all or part ofone or more T epitope(s) of the proteins produced at the hepatic stageof P. falciparum, characterized in that this peptide sequence comprisessuccessively:

-   -   all or part of the sequence of the first 153 amino acids shown        in FIGS. 7A–7C,    -   optionally, all or part of one or more of the sequences of 17        amino acids (SEQ ID NOS: 2–18) of formula:

X₁DLEQX₂RX₃AKEKLQX₄QQ QX₁DLEQX₂RX₃AKEKLQX₄Q QQX₁DLEQX₂RX₃AKEKLQX₄X₄QQX₁DLEQX₂RX₃AKEKLQ QX₄QQX₁DLEQX₂RX₃AKEKL LQX₄QQX₁DLEQX₂RX₃AKEKKLQX₄QQX₁DLEQX₂RX₃AKE EKLQX₄QQX₁DLEQX₂RX₃AK KEKLQX₄QQX₁DLEQX₂RX₃AAKEKLQX₄QQX₁DLEQX₂RX₃ X₃AKEKLQX₄QQX₁DLEQX₂R RX₃AKEKLQX₄QQX₁DLEQX₂X₂RX₃AKEKLQX₄QQX₁DLEQ QX₂RX₃AKEKLQX₄QQX₁DLE EQX₂RX₃AKEKLQX₄QQX₁DLLEQX₂RX₃AKEKLQX₄QQX₁D DLEQX₂RX₃AKEKLQX₄QQX₁in which:

-   -   X₁ is “Ser” or “Arg”,    -   X₂ is “Glu” or “Asp”    -   X₃ is “Arg” or “Leu”    -   X₄ is “Glu” or “Gly”        and all or part of the last 279 amino acids shown in FIGS.        10A–10D.

The invention also relates to any polypeptide composition constituted byseveral different peptide sequences which bear all or part of one ormore epitope(s) characteristic of a protein produced in the hepatocytesinfected by P. falciparum, as described above.

Generally speaking, by all or part of a peptide sequence of theinvention is meant any sequence comprising at least 4 to 5 amino acidsup to the maximal number of amino acids of the sequences describedabove.

It will be obvious that the free reactive functions that some aminoacids included in the composition of the molecules according to theinvention may possess, in particular the free carboxyl groups borne bythe Glu residues or by the C-terminal amino acid, on the one hand,and/or the free groups borne by the N-terminal amino acid or by aminoacids within the peptide chain, for example Lys, on the other, may bemodified, provided that this modification does not lead to amodification of the antigenic properties, or, according tocircumstances, immunogenic properties of the whole molecule. Themolecules thus modified are automatically included in the framework ofprotection given to the invention by the claims. These carboxylfunctions may possibly be acylated or esterified.

Other modifications are also included in the framework of the invention.In particular, the amine or ester functions of the terminal amino acids,or both at once, may themselves be linked to other amino acids. Forexample, the N-terminal amino acid may be linked to a sequencecomprising from one to several amino acids correponding to a part of theC-terminal region of another peptide conforming to the definition whichwas given to it above, or vice versa.

It will also be obvious that any peptide sequence derived from themodification by substitution and/or by addition and/or deletion of oneor more amino acids of one of the peptide sequences described above isincluded in the framework of protection given to the invention by theclaims, provided that this modification does not impair the antigenic orimmunogenic properties of the polypeptides of the invention, inparticular when these immunogenic properties have been reinforcedadequately, for example by combination of these polypeptides with asuitable immunological adjuvant (for example, a muramyl peptide) or bycoupling to a carrier molecule of higher molecular weight (for example,a serum albumin or a polylysine) or a toxin of the tetanus type oranother antigen of P. falciparum.

More particularly, the invention relates to any peptide sequence derivedfrom the peptide sequences mentioned above, and exhibiting modificationsresulting from substitution of maximally 40% of the amino acids whileretaining the biological activity of the sequences of the invention,namely in particular the induction of a response of the T lymphocytes,in particular the cytotoxic T lymphocytes.

The invention relates more generally to any molecule characterized bythe presence in its structure of one or more peptide sequences whichexhibit immunological cross-reactions with the peptide sequencescorresponding to the preceding formulae towards antibodies which can beinduced by these latter in vivo.

The invention also relates to any sequence of nucleotides which codesfor a polypeptide of the invention and, more particularly, any sequenceof nucleotides corresponding to one of the amino acid sequences of theinvention according to the universal genetic code.

The subject of the invention is more particularly the nucleotidesequence constituted by the 951 nucleotides shown in FIG. 2, and whichcode for the above-mentioned polypeptide of 316 amino acids (alsodesignated hereafter by the recombinant protein LSA-R-NR) shown in FIG.1.

The invention also relates to the nucleotide sequence shown in FIG. 8,which corresponds to the 3′ end of the LSA gene.

The invention also relates to the nucleotide sequences which code forpeptide subsequences of the invention. Particular mention should be madeof the nucleotide sequences delimited by the nucleotides situated at thepositions 630 to 949, 597 to 648 (coding for the peptide LSA-J) or 640to 717 (coding for the peptide LSA-NR) of FIG. 2.

The invention also relates to all or part of the nucleotide sequence ofFIG. 4 which codes for all or part of the peptide sequence 729S shown inFIG. 3.

The subject of the invention is also the nucleotide sequence shown inFIG. 5, which corresponds to the 5′ end of the LSA gene.

The invention also relates to any sequence of nucleotides which codesfor a polypeptide identical with, or one similar from the point of viewof both structure and antigenic properties to, those of the invention,this sequence being capable of hybridizing with all or part of thenucleotide sequence defined by the nucleotides situated at the positions597 to 949 of FIG. 2, or with all or part of the nucleotide sequence ofFIG. 4 or the sequences complementary to these latter, under thefollowing conditions:

-   -   pre-treatment (pre-hybridization) of the nitrocellulose filter        supporting the nucleic acid fragment to be tested with        hybridization buffer (composed of 6 SSC, 5× Denhardt's, 0.5%        SDS, 100 μg/l denatured, sonicated salmon sperm DNA) this        operation being carried Out at 65° C. for 1 hour;    -   replacement of the hybridization buffer in contact with the        support to which the nucleic acid fragment is now bound by        hybridization buffer of the same composition and addition of the        above-mentioned sequence shown in FIG. 2 (SEQ ID NO: 32) or FIG.        4 (SEQ ID NO: 33) as probe, in particular radioactively        labelled, and denatured beforehand;    -   incubation of the said nucleic acid fragment bound to the        support in this incubation buffer with the above-mentioned        sequence shown in FIG. 2 (SEQ ID NO: 32) or FIG. 4 (SEQ ID        NO: 33) at 65° C. for a period of about 1 hour;    -   the removal of the buffer containing the probe not bound by two        successive washings of 30 minutes each with a buffer solution        composed of 2×SSC and 0.5% SDS at 65° C.

It should be recalled that 20×SSC=175.3 g NaCl, 88.2 g trisodiumcitrate/l, pH 7; Denhardt's 50×=5 g Ficoll 400, 5 g polyvinylpyrrolidone, 5 g BSA (bovine serum albumin) fraction V/1; SDS is sodiumdodecyl sulfate.

The subject of the invention is any recombinant nucleic acid containingat least one nucleotide sequence of the invention inserted into anucleic acid heterologous with respect to the said nucleotide sequence.

The invention relates more particularly to a recombinant nucleic acidsuch as that defined above in which the nucleotide sequence of theinvention is preceded by a promoter (in particular, an induciblepromoter) under the control of which transcription of the said sequenceis capable of being carried out and optionally followed by a sequencecoding for signals for the termination of transcription.

The invention relates to any recombinant vector, used in particular forcloning a nucleotide sequence of the invention and/or the expression ofthe polypeptide encoded in this sequence, and characterized in that itcontains a recombinant nucleic acid such as that defined above at one ofits sites inessential for its replication.

As an example of a vector mentioned above, mention should be made ofplasmids, cosmids or phages.

Consequently, the invention relates more particularly to the plasmidDG536 deposited with the CNCM under the No. I-1027 on 17 Jan. 1991, aswell as the plasmid DG729S deposited with the CNCM under the No. I-1028on 17 Jan. 1991.

The subject of the invention is also a procedure for the preparation ofa polypeptide of the invention by transformation of a cell host with theaid of a recombinant vector of the type indicated above, followed by theplacing of the thus transformed cell host in culture and the recovery ofthe polypeptide from the culture medium.

Thus, the invention relates to any cell host transformed by arecombinant vector as defined above and which comprises the regulatoryelements which allow the expression of the nucleotide sequence codingfor a polypeptide according to the invention.

The subject of the invention is more particularly DNA (or RNA) primerswhich can be used in the context of the synthesis of nucleotide and/orpolypeptide sequences according to the invention by the PCR (PolymeraseChain Reaction) procedure, as described in the U.S. Pat. Nos. 4,683,202and 4,683,195 and the European patent application No. 200.362(PCR=assembly-line amplification of the DNA).

The invention relates to any DNA or RNA primer, characterized in that itis constituted of about 10 to 25 nucleotides identical with the first 10to 25 nucleotides of the nucleotide sequence which codes for a peptidesequence according to the invention or identical with the last 10 to 25nucleotides of the said sequence.

The invention also relates to any DNA or RNA primer, characterized inthat it is constituted of about 10 to 25 nucleotides complementary tothe first 10 to 25 nucleotides of the nucleotide sequence according tothe invention or complementary to the last 10 to 25 nucleotides of thesaid nucleotide sequence.

The subject of the invention is also any DNA or RNA primer,characterized in that it is constituted of about 10 to 25 nucleotideswhich are capable of hybridizing with the first 10 to 25 nucleotides orwith the last 10 to 25 nucleotides of the said nucleotide sequence whichcodes for a polypeptide of the invention under the conditions ofhybridization defined above.

Thus the present invention relates more particularly to a procedure forthe preparation of a polypeptide of the invention comprising thefollowing steps:

-   -   optionally, the prior amplification according to the PCR        procedure of a quantity of the nucleotide sequence which codes        for the said polypeptide with the aid of two DNA primers        selected such that one of these primers is identical with the        first 10 to 25 nucleotides of the nucleotide sequence which        codes for the said polypeptide, whereas the other primer is        complementary to the last 10 to 25 nucleotides (or hybridizes        with these last 10 to 25 nucleotides) of the said nucleotide        sequence, or conversely such that one of these primers is        identical with the last 10 to 25 nucleotides of the said        sequence, whereas the other primer is complementary to the first        10 to 25 nucleotides (or hybridizes with these first 10 to 25        nucleotides) of the said nucleotide sequence, followed by the        introduction of the said nucleotide sequences thus amplified        into a suitable vector,    -   the placing in culture in a suitable culture medium of a cell        host previously transformed by a suitable vector containing a        nucleic acid according to the invention containing the        nucleotide sequence which codes for the said polypeptide, and    -   the recovery of the polypeptide produced by the said transformed        cell host from the above-mentioned culture medium.

As examples of DNA or RNA primers according to the invention, mentionshould be made of the following sequences (SEQ ID NOS: 29 and 30):

-   -   3′→5: TTTCGCTAGATCTTGTT & TCTAAATAGAAGAAA

The peptides according to the invention may also be prepared by thestandard procedures used in the field of peptide synthesis. Suchsynthesis may be carried out in homogeneous solution or on a solid phase

For example, recourse may be had to the synthetic procedure inhomogeneous solution described by HOUBENWEYL in the monograph entitled“Methoden der Organischen Chemie” (Methods of Organic Chemistry) editedby E. Wunsch, vol. 15-I and II, THIEME, Stuttgart 1974.

This method of synthesis consists of successively condensing thesuccessive aminoacyl residues in the required order, or of condensingaminoacyl residues and fragments previously formed which already containseveral amino acids in the correct order, or also of condensing severalfragments thus prepared beforehand, it being understood that care willbe taken to protect beforehand all of the reactive functions borne bythese aminoacyl residues or fragments, with the exception of the aminofunction of the one and the carboxyl function of the other or viceversa, which are usually required to participate in the formation of thepeptide bonds, in particular after activation of the carboxyl functionaccording to the methods well known in peptide synthesis. As a variant,it will also be possible to have recourse to coupling reactions whichmake use of standard coupling reagents of the carbodiimide type, such asfor example 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

When the amino acyl residue used possesses an additional acidic function(in particular in the case of glutamic acid), such functions should beprotected for example by t-butyl ester groups.

In the case of stepwise synthesis, the amino acids being added one at atime, the synthesis begins preferably with the condensation of theC-terminal amino acid with the amino acid which corresponds to theneighbouring aminoacyl residue in the desired sequence and so on, oneafter the other, until the N-terminal amino acid is reached.

According to another preferred procedure of the invention, recourse ishad to that described by R. B. MERRIFIELD in the article entitled “Solidphase peptide synthesis” (J. Am. Chem. Soc., 45, 2149–2154).

In order to synthesize a peptide chain according to the MERRIFIELDprocedure, recourse is had to a very porous polymeric resin to which thefirst, C-terminal amino acid of the chain is attached. This amino acidis attached to the resin through the intermediary of its carboxyl groupand its amino function is protected, for example by means of thet-butoxycarbonyl group.

When the first, C-terminal amino acid has thus been attached to theresin, the protecting group of the amino function is removed by washingthe resin with acid.

In the case in which the protecting group of the amine function is thet-butoxycarbonyl group, it may be removed by treatment of the resin withtrifluoroacetic acid.

The second amino acid is then coupled to the deprotected amine functionof the first C-terminal amino acid to furnish the second aminoacylresidue of the desired sequence, counting from the C-terminus.Preferably, the carboxyl function of this second amino acid is activatedfor example by means of dicyclohexylcarbodiimide and the amine functionis protected, for example by means of t-butoxycarbonyl.

The first part of the desired peptide chain is thus produced, whichcontains two amino acids and the terminal amino function of which isprotected. As previously, the amine function is deprotected and it isthen possible to proceed to the attachment of the third aminoacylresidue under conditions analogous to those for the addition of thesecond, penultimate C-terminal amino acid.

In this way, the amino acids which will constitute the peptide chain areadded one after the other to the previously deprotected amine group ofthe portion of the peptide chain already formed which is attached to theresin.

When the desired peptide chain has been assembled in its entirety, theprotecting groups of the different side chains of the amino acidsconstituting the peptide chain are removed and the peptide is cleavedfrom the resin, for example with the aid of hydrogen fluoride.

The invention also relates to water-soluble oligomers of the monomericpeptides indicated above.

The oligomerization may cause an increase in the immunogenicity of themonomeric peptides according to the invention. Without such numericalvalues being considered as limiting, it should nonetheless be mentionedthat these oligomers may contain, for example, from 2 to 10 monomericunits.

In order to carry out the oligomerization, recourse may be had to anypolymerization procedure commonly used in the field of peptides, thispolymerization being conducted until an oligomer or polymer is obtainedwhich contains the required number of monomeric motifs for theacquisition of the desired immunogenicity.

One method of oligomerization or polymerization of the monomer consistsin the reaction of the latter with a cross-linking agent such asglutaraldehyde.

It is also possible to have recourse to other methods of oligomerizationor coupling, for example to that making use of the successive couplingof monomeric units through the intermediary of their terminal carboxyland amino functions in the presence of homo- or hetero-bifunctionalcoupling agents.

The invention also relates to the conjugates obtained by covalentcoupling of the peptides according to the invention (or theabove-mentioned oligomers) to carrier molecules (natural or synthetic),physiologically acceptable and non-toxic, through the intermediary ofcomplementary reactive groups borne respectively by the carrier moleculeand the peptide. Examples of suitable groups are illustrated in whatfollows:

As examples of carrier molecules or macromolecular supports forming partof the composition of the conjugates according to the invention, mentionshould be made of naturally occurring proteins such as tetanus toxoid,ovalbumin, serum albumin, hemocyanins, the PPD of tuberculin (PPD:“Purified Protein Derivative”), etc . . .

Mention should be made, for example, of polylysines or poly(D-L-alanine)-poly (L-lysine) as examples of synthetic macromolecularsupports.

The literature mentions other types of macromolecular supports which canbe used and which usually have a molecular weight higher than 20,000.

In order to synthesize the conjugates according to the invention,recourse may be had to known procedures such as that described by FRANTZand ROBERTSON in Infect. and Immunity, 33, 193–198 (1981) or thatdescribed in Applied and Environmental Microbiology, (October 1981),vol. 42, No. 4, 611–614 by P. E. KAUFFMAN by using the peptide and theappropriate carrier molecule.

In practice, the following compounds, cited in a non-limiting manner,are advantageously used as coupling agents: glutaraldehyde, ethylchloroformate, water-soluble carbodiimides : N-ethyl-N′(3-dimethylamino-propyl) carbodiimide HCl , diisocyanates,bis-diazobenzidine, di- and tri-chloro-s-triazines, cyanogen bromide aswell as the coupling agents mentioned in Scand. J. Immunol., (1978),vol. 8, p. 7–23 (AVRAMEAS, TERNYNCK, GUESDON).

It is possible to have recourse to any coupling procedure implicating,on the one hand, one or more reactive functions of the peptide and, onthe other, one or more reactive functions of the molecular supports.Advantageously, these are carboxyl and amine functions which can giverise to a coupling reaction in the presence of a coupling agent of thetype used in the synthesis of proteins, for example,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxybenzotriazole,etc. . . . It is also possible to have recourse to glutaraldehyde, inparticular when it is required to link together amino groups borne bythe peptide and the molecular support, respectively.

The nucleic acids of the invention can be prepared either by a chemicalprocedure or by other procedures.

A suitable method of preparation of the nucleic acids containing amaximum of 200 nucleotides (or 200 bp when double-stranded nucleic acidsare concerned) of the invention comprises the following steps:

-   -   the synthesis of DNA using the automated        beta-cyanoethylphosphoramidite method described in Bio-organic        Chemistry 4; 274–325 (1986),    -   the cloning of the nucleic acids thus obtained in a suitable        vector and the recovery of the nucleic acid with a suitable        probe.

A method of preparation by the chemical route of nucleic acids longerthan 200 nucleotides (or 200 bp when double-stranded nucleic acids areconcerned) of the invention comprises the following steps:

-   -   the assembly of chemically synthesized oligonucleotides,        provided at their ends with different restriction sites, the        sequences of which are compatible with the amino acid sequence        of the natural polypeptide according to the principle described        in Proc. Natl. Acad. Sci. USA, 80; 7461–7465, (1983),    -   the cloning of the nucleic acids thus obtained in a suitable        vector and the recovery of the desired nucleic acid by means of        hybridization with a suitable probe.

The nucleic acids of the invention can also be prepared in the followingmanner:

-   -   incubation of the genomic DNA isolated from a strain of P.        falciparum with DNase I, then addition of EDTA and purification        by extraction with the mixture phenol/chloroform/isoamyl alcohol        (25/24/1), then with ether,    -   treatment of the DNA thus extracted with the EcoRI methylase in        the presence of DTT, and purification by extraction as described        above,    -   incubation of the DNA thus purified with the 4 deoxynucleoside        triphosphates dATP, dCTP, DGTP and dTTP in the presence of T4        DNA polymerase and DNA ligase of E. coli, followed by        purification according to the method described above,    -   the cloning of the nucleic acids thus obtained in a suitable        vector and the recovery of the desired nucleic acid with the aid        of a suitable probe.

The nucleotide probes used for the recovery of the desired nucleic acidin the procedures mentioned above are usually composed of 40 to 200nucleotides of the nucleotide sequence shown in FIG. 2 (selected moreparticularly from those situated between the positions 597 to 949 shownin FIG. 2) or in FIG. 4 or its complementary sequence, and are capableof hybridizing with the desired nucleic acid under the conditions ofhybridization defined above. The synthesis of these probes is carriedout according to the automated beta-cyanoethylphosphoramidite methoddescribed in Bio-organic Chemistry 4, 274–325 (1986).

The molecules according to the invention possess antigenic propertiescharacteristic of the antigens which bear T epitopes, and optionally Bepitopes, and which are either specific for the hepatic stage of thedevelopment of P. falciparum or specific for the sporozoite, hepatic andblood stages, simultaneously.

In fact, as will be described more particularly with the aid of examplesof molecules according to the invention in the detailed descriptionwhich follows, the molecules according to the invention which containall or part of the amino acid sequence comprised between the positions200 and 316 shown in FIG. 1 (SEQ ID NO: 31), react specifically with theantibodies or the lymphocytes directed against the B and/or T epitopesof the antigens produced at the hepatic stage of P. falciparum, but notwith the antibodies directed against other antigens produced by P.falciparum, or against antigens produced by other species of Plasmodium.

These molecules according to the invention thus recognize specificallythe antibodies produced by the immune system of an individual infectedby P. falciparum under the influence of the LSA antigen, the stronglyimmunogenic character of which has already been mentioned.

These molecules according to the invention comprising all or part of thepeptide sequence shown in FIG. 3 (SEQ ID NO: 24) are not recognized bythe former antibodies which react specifically with all or part of thepolypeptide defined by the amino acids situated at the positions 200 to316 in FIG. 1.

On the other hand, the polypeptides corresponding to all or part of thepeptide sequence shown in FIG. 3 (SEQ ID NO: 24) are recognized byantibodies which react specifically with antigens localized on thesurface of sporozoites (derived from different strains of P. falciparum)as well as with antigens of the hepatic schizonts and the bloodschizonts, and finally with the surface of the sporozoites of P. yoeliibut not of P. berghei.

It should also be emphasized that the antibodies which recognizespecifically the polypeptides corresponding to all or part of thepeptide sequence shown in FIG. 3 (SEQ ID NO: 24) are capable of blockingcompletely the entry of the sporozoites of P. yoelii into hepatic cellsof rodents in vitro, unlike the antibodies directed against thecircumsporozoite protein of P. yoelii and of P. falciparum.

The possibility of producing molecules according to the invention inlarge amounts as well as their properties of specific recognition ofantibodies included among the most actively produced on infection of anindividual by P. falciparum make the said molecules the reagents ofchoice for the in vitro diagnosis of malaria in an individual infectedby P. falciparum.

The invention thus relates to a procedure for the in vitro detection ofantibodies which correlate with malaria originating from the infectionof an individual by P. falciparum in a tissue or biological fluid likelyto contain them, this procedure comprising the placing of this tissue orbiological fluid in contact with a molecule according to the inventionunder conditions which allow an in vitro immunological reaction betweenthe said molecules and the antibodies possibly present in the tissue orbiological fluid to occur, and the in vitro detection of theantigen-antibody complexes possibly formed.

The biological fluid is preferably constituted by a human serum.

Any standard procedure may be used to carry out such a detection.

As an example, a preferred method makes use of immunoenzymatic processesaccording to the ELISA procedure, or immunofluorescent orradioimmunological (RIA) or equivalent procedures.

Thus, the invention also relates to any molecule according to theinvention labelled with the aid of a suitable label of the enzymatic,fluorescent, radioactive, etc . . . type.

Such methods comprise for example the following steps:

-   -   the loading of defined quantities of a polypeptide composition        according to the invention into the wells of a microtitration        plate,    -   introduction into the said wells of increasing dilutions of        serum requiring diagnosis,    -   incubation of the microplate,    -   repeated rinsings of the microplate,    -   introduction into the wells of the microplate labelled        antibodies against the immunoglobulins of the blood, the        labelling of these antibodies having been carried out with the        aid of an enzyme selected from those which are capable of        hydrolysing a substrate and of thus changing the absorption of        the latter, at least at one particular wavelength,    -   detection of the quantity of substrate hydrolysed, in comparison        with a control.

The invention also relates to kits for the in vitro diagnosis of malariacaused by P. falciparum which contain:

-   -   a polypeptide composition according to the invention,    -   the reagents for the constitution of the medium suitable for        carrying out the immunological reaction,    -   the reagents necessary for the detection of the antigen-antibody        complexes produced by the immunological reaction, these reagents        may also be labelled, or be capable of being recognized in turn        by a labelled reagent, more particularly in the case in which        the above-mentioned polypeptide composition is not labelled.    -   a reference tissue or biological fluid lacking antibodies        recognized by the above-mentioned polypeptide composition.

The invention relates to the antibodies themselves formed against thepolypeptides of the invention.

It will be obvious that these antibodies are not limited to polyclonalantibodies.

The invention also applies to any monoclonal antibody produced by anyhybridoma capable of being formed by standard methods from the spleencells of an animal, in particular a mouse or a rat, immunised againstone of the purified peptides of the invention, on the one hand, andcells of a suitable myeloma cell line, on the other, and which can beselected for its capacity to produce monoclonal antibodies whichrecognize the polypeptide initially used for the immunization of theanimals.

The invention also relates to a method for the in vitro diagnosis ofmalaria in an individual likely to be infected by P. falciparum whichcomprises the placing of a tissue or biological fluid taken from anindividual in contact with antibodies such as those described aboveunder conditions which allow an in vitro immunological reaction betweenthe said antibodies and the proteins specific for P. falciparum possiblypresent in the biological tissue to occur, and the in vitro detection ofthe antigen-antibody complexes possibly formed.

Consequently, the subject of the invention is a kit for the in vitrodiagnosis of malaria containing:

-   -   antibodies such as those described above,    -   the reagents for making up the appropriate medium for carrying        out the immunological reaction,    -   the reagents making possible the detection of the        antigen-antibody complexes produced by the immunological        reaction, these reagents may also be labelled, or be capable of        being recognized in turn by a labelled reagent, more        particularly in the case in which the above-mentioned        polypeptide composition is not labelled.

The invention also relates to a nucleotide detection probe characterizedin that it is composed of all or part of one of the nucleotide sequencesof the invention such as defined above.

More particularly the subject of the invention is an in vitro diagnosticmethod for malaria in an individual likely to be infected by P.falciparum which comprises the following steps:

-   -   possibly prior amplification of the quantity of nucleotide        sequences according to the invention likely to be contained in        the biological sample taken from the said individual, with the        aid of two DNA primers selected in the manner indicated above,    -   the placing of the above-mentioned biological sample in contact        with a nucleotide probe such as that defined above under        conditions which allow he production of a hybridization complex        formed between the said probe and the said nucleotide sequence,    -   the detection of the above-mentioned hybridization complex        possibly formed.

As examples of nucleotide probes of the invention, mention should bemade of the following sequences:

3′→5═:TTTCGCTAGCTCTTGTT & TCTAAATAGAAGAAA

Most especially, the invention opens the door to the development ofnovel vaccinating principles against malaria originating from theinfection of an individual by P. falciparum.

The invention also relates to the compositions prepared in the form ofvaccines which contain either one or more peptides according to theinvention or an oligomer of this or these peptide(s) or also a conjugateof this or these peptide(s) or oligomer with a carrier molecule, incombination with a suitable pharmaceutically acceptable vehicle and,optionally, with 5other active ingredients which vaccinate againstmalaria.

A particularly useful pharmaceutical composition of the invention ischaracterized in that it contains all or part of the peptide sequencedefined by the amino acids situated at the positions 200 to 316 of FIG.1, in combination with all or part of the peptide sequence shown in FIG.3.

Advantageous pharmaceutical compositions are constituted by solutions,suspensions or injectable liposomes containing an efficacious dose of atleast one product according to the invention. Preferably, thesesolutions, suspensions or liposomes are prepared in an isotonicsterilized aqueous phase, preferably a saline or glucose solution.

The invention relates more particularly to such suspensions, solutionsor liposomes which can be administered by intradermal, intramuscular orsubcutaneous injection or even by scarification.

It also relates to pharmaceutical compositions which can be administeredby other routes, in particular the oral or rectal route, or also in theform of aerosols designed to come into contact with mucous membranes, inparticular the ocular, nasal, pulmonary or vaginal mucous membranes.

Consequently, it relates to pharmaceutical compositions in which atleast one of the products according to the invention is combined withpharmaceutically acceptable excipients, solid or liquid, suited to thecomposition of oral, ocular or nasal forms, or with excipients suited tothe composition of the rectal forms of administration, or also withgelatinous excipients for vaginal administration. It also relates toisotonic liquid compositions containing at least one of the conjugatesaccording to the invention, adapted to administration to mucousmembranes, in particular ocular or nasal mucous membranes.

Advantageously, the vaccinating compositions according to the inventioncontain in addition a vehicle such as polyvinyl-pyrrolidone, whichfacilitates the administration of the vaccine. Instead ofpolyvinyl-pyrrolidone it is possible to use any other type of adjuvantin the classical sense which this expression used to connote, i.e. asubstance which makes the absorption of the medicine easier orfacilitates its action in the organism. As examples of other adjuvantsof this latter type, mention should also be made ofcarboxymethylcellulose, the hydroxides and phosphates of aluminium,saponin or all other adjuvants of this type, well known to thespecialist skilled in the art. Finally, if necessary, they contain animmunological adjuvant, in particular of the muramyl peptide type.

The invention also relates to pharmaceutical compositions containing asactive substance at least one of the monoclonal or polyclonal antibodiespreviously defined in combination with a pharmaceutically acceptablevehicle.

The invention is obviously not limited to the embodiments describedabove as examples and the specialist skilled in the art may makemodifications to them without exceeding the limits of the framework ofthe claims given hereafter; in particular, some of the amino acids ofthe sequence of the peptides according to the invention may be replacedby isofunctional or isosteric amino acids; for example, one or more ofthe following substitutions may be envisaged:

-   -   Glu is replaced by Asp or Gln,    -   Leu is replaced by Ala, etc.

It is naturally understood that the peptides which result from suchsubstitutions consist of equivalents of the peptides more particularlyclaimed, provided that they themselves or oligomers or conjugates formedfrom these peptides exhibit similar immunogenic properties.

The invention also relates more particularly to the “chimeric proteins”which can be obtained by the procedures of genetic engineering, thesechimeric proteins containing one or more of the peptide sequences of theinvention, and being incorporated into or attached to a peptide fragmentother than beta-galactosidase. This latter peptide fragment preferablyhas a molecular weight sufficient to reinforce the immunogenicity of thepeptide sequences according to the invention and does not interfereimmunologically with the expression of the desired immunogenicity.

Additional characteristics of the invention will also become apparent inthe course of the description which follows of the conditions underwhich the polypeptides of the invention were obtained.

Sera derived from Europeans living in endemic areas and following acontinuous prophylaxis with medicines directed against the schizonts ofthe blood stages (chloroquine) were selected and tested by usingantigens of the sporozoite stage, the hepatic stage (LS antigen) and theblood stages. Most of these sera react with the antigens of all of thestages probably because the prophylaxis had been interrupted. Three serataken from individuals who had resided in rural tropical Africa and whohad ingested 100 ug of chloroquine per day without interruption for 23to 26 years did not react with the antigens of the blood stagesaccording to the immunofluorescence assay (IFA). However, these threesera possessed high titers of antibodies directed against thesporozoites and the LSA proteins (dilution IFA 1/3200 and 1/6400,respectively).

One of the three sera just mentioned, with reduced specificity, was usedto screen a genomic DNA library constructed in the bacteriophage λ gt 11in the following manner:

-   1) CONSTRUCTION OF THE GENOMIC DNA LIBRARY OF Plasmodium falciparum.

The genomic DNA of clone 96 of the Thailand Tak9 strain of P. falciparum(Science, 212, 137–138 (1981) was isolated by standard procedures.

Samples of 18 μg of DNA of P. falciparum were incubated at 15° C. in a50 mM Tris HCl buffer, pH 7.5, 1 mM MnCl₂, 20 μg/ml of bovine serumalbumin with variable amounts of DNAase I (Boehringer Mannheim): 5 pgfor 5 minutes or 3.5 pg for 5 or 10 minutes. After addition of 5 mM EDTA(ethylenediamine tetraacetic acid), the samples of DNA are pooled andpurified by extraction with a phenol/chloroform/isoamyl alcohol mixture(25 V/24 V/1 V), then with ether. The DNA is concentrated byprecipitation with ethanol at −20° C. in the presence of 2.5 M ammoniumacetate.

45 μg of DNA thus treated was methylated by means of 180 U of EcoR1methylase (Biolabs) under the conditions recommended by the supplier,with the further addition of 5 mM DTT (dithiothreitol) for 15 minutes at37° C. After purification of the DNA as above, 10 ug of DNA wereincubated with 40 mM Tris HCl, pH 8.0, 10 mM ammonium sulfate, 10 mM2-mercaptoethanol, 0.5 mM EDTA, 0.05 mM NAD (nicotinamide adeninedinucleotide) 0.1 mM dXTP (comprising the 4 deoxynucleosidestriphosphates dATP, dCTP, dGTP and dTTP) in the presence of 10 U T4 DNApolymerase (PL Biochemicals) and 10 U of E. coli DNA ligase (Biolabs).The DNA was purified and concentrated as above.

8 μg of DNA were then ligated with 0.4 ug of an EcoR1 adaptor or“linker” (EcoR1 phosphorylated adaptor marketed by Biolabs) by means of4 U T4 DNA ligase (Biotec) in a 50 mM Tris HCl buffer, pH 8.0, 10 mMMgCl₂, 20 mM DTT, 1 mM ATP, 50 μg/ml bovine serum albumin.

After incubation at 4° C. for 5 hours, 2 U T4 DNA ligase are added andthe reaction is allowed to proceed at 4° C. for 16 hours. The tube issubjected to several cycles of freezing to −80° C./thawing to stop thereaction. The DNA is then diluted and the incubation buffer is adjustedso as to produce the conditions recommended by the supplier for the useof the enzyme EcoR1. 100 U of enzyme EcoR1(Promega Biotec) are added andincubated for 3 hours at 37° C. The reaction is stopped by heating for10 minutes at 60° C., and the DNA is purified and concentrated as above.

The DNA is resuspended in 100 μl of 50 mM Tris HCl buffer, pH 8.0, 1 mMEDTA and loaded on to a 5–20% sucrose gradient prepared in 25 mM sodiumacetate, 10 mM EDTA and centrifuged in the Beckmann rotor SW 50.1 at45,000 revolutions per minute for 150 minutes.

The fractions are analysed on agarose gel and those which contain theDNA fragments of a size included between about 300 bp and 2,500 bp arepooled, dialysed against 50 mM Tris HCl buffer, pH 8.0, 1 mM EDTA at 4°C. The DNA is concentrated by precipitation with ethanol. About 400 ngof this DNA were ligated to 1 μg of DNA of the vector λgt11 (Proc. Natl.Acad. Sci., USA, 80, 1194–1198 (1983) ) cut with EcoR1 anddephosphorylated (Protoclone from Promega Biotec) in a volume of 10 μl(in 50 mM Tris HCl buffer, pH 8.0, 10 mM MgCl₂, 20 MM DTT, 1 mM ATP, 50μg/ml bovine serum albumin) by 1 U T4 DNA ligase (Biotec).

The ligation products were encapsidated in vitro in E. coli extractsprepared from the bacterial strains constructed by B. Hohn (MethodsEnzymol. 68, 299) according to the procedure described by Maniatis etal. (Molecular cloning, a laboratory manual, p. 264, Cold Spring HarborLaboratory (1982)).

About 7 millions of recombinant bacteriophages were obtained.

-   2) IMMUNOLOGICAL SCREENING OF THE BANK

The recombinant bacteriophages were spread on a culture mediumcontaining the indicator bacteria Y 1090 at a density of 50,000 plaquesper 90 mm Petri dish, and incubated at 42° C. for 3 hours. Anitrocellulose filter (Schleicher & Schuell, BA 85) saturated with 0.01M IPTG isopropyl-beta-thiogalactopyranoside (Sigma) is deposited on thedishes which are incubated at 37° C. for 3 hours. When the incubationsare complete, the nitrocellulose filters are removed and the Petridishes are stored at 4° C.

The nitrocellulose filters are placed in a bath of TL buffer: 50 mM TrisHCl, pH 8.0, 150 mM NaCl, 5% skimmed milk, 0.05% Tween 20 (Sigma). Thefilters are incubated for 15 hours at 4° C. in TL buffer, then twice for15 minutes at 20° C. They are then incubated for one hour with a pool ofhuman immune antisera directed against the antigens of all stages ofdevelopment of P. falciparum, treated beforehand to deplete it ofanti-E. coli antibodies according to the procedure described by Ozaki etal. (J. Immunol. Methods, 89, 213–219, 1986). The pool of human antiserawas used at a dilution of 1/200 in TL buffer. The incubation was carriedout at 20° C. for 1 hour. The filters were washed 4 times with the TLbuffer, then incubated with anti-human immunoglobulin antibodiesconjugated to horseradish peroxidase (Biosys) and iodinated with ¹²⁵Ifor 1 hour at 20° C. After several washings with TL buffer, followed by50 mM Tris HCl buffer, pH 8.0, 150 mM NaCl, the enzymatic activity ofthe peroxidase is revealed (Ozaki et al., previously cited), the filtersare dried in air and autoradiographied using Kodak film Royal X-OMat AR,with an amplifying screen.

A collection of about 1200 clones of recombinant bacteriophages wasestablished by selecting the lysis plaques corresponding to the positivesignals. These clones were then subjected to a second cycle ofimmunological screening by using this time one of the three human serapreviously described and exhibiting few or no antibodies directedagainst the erythrocytic forms of P. falciparum and a high titer againstthe sporozoite and hepatic forms of the parasite. This immunologicalscreening was carried out according to the protocol described above.This serum has led to the identification of about recombinant 120 clonesout of 1200 tested.

The human antibodies which react with the antigenic determinantsexpressed by the recombinant clones were purified by means of theiraffinity for the recombinant proteins according to the proceduredescribed by Ozaki et al. (previously cited). These specific antibodieswere incubated with preparations of parasites at different stages ofdevelopment (sporozoite, hepatic stage or erythrocytic stages), and thereaction was studied by means of indirect immunofluorescence.

The recombinant clones on which the antibodies specific for the hepaticstage are retained by affinity and which thus express determinantsintrinsic to this stage were studied: they are the clones DG 307, DG 199and DG 145. These specific antibodies of these three clones reactspecifically with the hepatic schizonts such as can be obtained afterinfection of human or monkey hepatocytes by sporozoites of P.falciparum; the localization of the fluorescence was determined to beidentical with that considered to be characteristic of LSA.

The species- and stage-specificity of the 3 clones DG 145, DG 199 and DG307 were tested in the following manner. Firstly, it was determined thatthe same antibodies purified by affinity and which react by IFA (or alsowhich are IFA-positive) with LSA do not react with dried or moistpreparations of sporozoites, nor with the antigens of the blood stages,whether they are tested by IFA with parasites fixed in acetone or byimmunoblotting by using proteins of all of the stages extracted withSDS. The antibodies purified by affinity do not react with the antigensof the hepatic stage of P. yoelii, nor with the hepatic schizonts of P.vivax prepared from Saimiri sciureus monkeys.

Secondly, the recombinant proteins of DG 145, DG 199 and DG 307 do notreact with the sera obtained from two patients suffering from malaria(caused by P. falciparum) by accidental transfusion and which, bydefinition, thus do not have antibodies against the antigens specificfor the earlier stages (sporozoites and antigens of the hepatic stage).These proteins do not react with two monoclonal antibodies whichrecognize the CS tetrapeptide, with the sera of mice immunised with therecombinant CS antigens R32t and 32 (Science, 228, 958 (1985) ).Furthermore, the recombinant proteins did not react with human antiseradirected against P. vivax (although the sera were positive with thehepatic schizonts of P. vivax, P. ovale and P. cynomolgi (Ann. Soc.Belg. Med. Trop., 60, 348 (1980) ) when they are tested by the techniqueof immunodot blots, whereas they are positive with all of the humananti-P. falciparum sera tested.

Within this sub-population of 120 clones mentioned above, animmunological screening procedure using antibodies purified by affinityto the clone DG307 (or 145, or 199) leads to the detection of about 40clones out of 120 apparently exhibiting the characteristic epitope ofthe LSA and defined by the basic structure of 17 amino acids citedabove.

Similarly, identification procedures based on hybridization with the aidof DNA fragments from the same clones (DG307) make it possible toidentify these repetitive structures in the same clones as thoseidentified by the immunological assays.

A complementary screening of the sub-population of 40 clones belongingto this family of the LSA antigen was used to identify other parts ofthe gene containing sequences distinct from those defined by the repeatsof 17 amino acids. For this complementary screening sera identified asnot reacting with the repeats of the 17 amino acids but positive inindirect immunofluorescence with the peripheral structure of the hepaticschizont in which the LSA antigen is situated, were employed. Within thefamily of the 40 clones of the LSA, these sera were found to be positivefor several of them, and one of the clones containing the largestinsert, designated DG536, was selected and studied in detail.

Other clones, DG538, DG750 and DG443 were studied. The clones DG750 andDG443 contain the major part of the non repetitive 5′ sequence of theLSA gene.

The insert of 951 base pairs was purified and recloned in thebacteriophage M13 mp19. The DNA sequence and the genomic organization ofthe LSA gene were then determined. FIG. 1 (SEQ ID NO: 31) shows that theclone contains a sequence of 209 amino acids at the 5′ end correspondingto a series of 12 repeats of 17 amino acids, similar to that describedin the article by Guérin-Marchand et al. (Nature, mentioned above) andthen contains a set of 106 amino acids, the structure of which is notrepetitive.

As can be seen in FIG. 1 (SEQ ID NO: 31), the motif of 17 amino acids isin two repeats (cf. motif corresponding to the positions 35 to 51, andthat corresponding to the positions 137 to 153 of FIG. 1) identical withthat described in the article by Guérin-Marchand et al. and the otherrepeats exhibit a substitution of a leucine by an arginine (cf.positions 8, 59, 76, 110, 127, 161, 178 and 195 of FIG. 1) (SEQ ID NO:31), a substitution of a glutamic acid by an aspartic acid (cf.positions 23 and 91 of FIG. 1 (SEQ ID NO: 31)) as well as a substitutionof a serine by an arginine (cf. position 205 of FIG. 1 (SEQ ID NO: 31)).

It was possible to measure the size of the native protein of the LSA inthe parasite after surmounting great difficulties. Hepatic schizonts ofP. falciparum were produced in vitro by infection of human hepatocytesfrom a primary culture with salivary glands of mosquitoes containingsporozoites according to the procedure described in Mazier et al.(Science No. 227, p440, 1985). After 7 days of culture, the infectedcells are recovered and used to prepare an extract analysed onpolyacrylamide gel containing SDS and transferred to nitrocellulose. Thehepatic antigens are then revealed by an antibody purified by affinityto the repeats of the 17 amino acids of the LSA already mentioned. Theseantibodies label a protein of molecular weight of 200,000 Daltons.

The hybridization of the clone DG307 with the DNA fragments of P.falciparum obtained by digestion with a restriction enzyme, the Mungbean nuclease, which under the conditions described by McCutchans (NAR,16, 14, 6883–6896, 1988) is capable of cutting each end of the genes ofthe parasite, reveals a unique band of molecular weight of 5 K basesconsistent with the size of the native protein measured in the parasiteextract.

In electron microscopy of the hepatic schizonts obtained by injection ofsporozoites of chimpanzees, the labelling of the anti-LSA antibodiespurified by affinity, visualized by a second antibody labelled withcolloidal gold, reveals that the LSA molecule is distributed: a) at thestage of the hepatic trophozoite and the young schizont, in vacuolescontaining granules which open and discharge their contents into theparasitophorous vacuole, b) at the stage of the 5 days old immatureschizont, at the periphery of the hepatic schizont in the granulespresent in the parasitophorous vacuole of the parasite, c) in the 6 to 7days old mature schizonts, in the parasitophorous vacuole as well asbetween the pseudo-cytomers of the schizont, then finally surroundingthe merozoites in process of formation.

The study of the immunological response of subjects exposed to malariaas well as animals immunized with the recombinant and/or syntheticproteins makes it possible to specify the biological function of the LSAprotein and of certain segments of this protein, in particular thosecontained in the synthetic peptides.

The immunization of mice with the LSA-R-NR proteins and the study of theresponse of the lymphocytes of these mice, as well as the immunizationwith the LSA-R peptides of mice of different haplotypes with peptidesLSA-R, LSA-J and LSA-NR, and finally the study of the responses of thelymphocytes of the subjects exposed to malaria towards the LSA-Rpeptides had shown that a T epitope for man and the mouse is not definedby the repetitive part of the LSA molecule. More detailed studies showthe existence of a T epitope in the LSA-R peptide. As had been shownpreviously, the LSA-R peptide constitutes an excellent B epitope for manand the mouse, defined by the repetitive part of the LSA molecule, andthe complementary results obtained since in more than 500 individualsexposed to malaria show that this epitope is recognized by theantibodies of about 95% of the subjects studied, in Senegal, UpperVolta, Madagascar and Kenya.

The preliminary study of the lymphocytes of 5 adult African subjectsexposed to malaria, subsequently confirmed by the detailed study of theresponse of the peripheral lymphocytes of more than 200 adult Africansubjects exposed to malaria as well as the lymphocytes of chimpanzees(Pan troglodytes) immunised by the recombinant protein LSA-R-NR (cloneDG536), revealed that a T epitope of the LSA molecule is defined by theamino acid sequence contained in the synthetic peptide LSA-NR. Two otherT epitopes are contained in the sequences of the synthetic peptidesLSA-J and LSA-R (in total 39% of positive responses to the T epitopes ofthe LSA in Madagascar and 83% in Senegal). Proliferative responses ofthe human lymphocytes and the lymphocytes of an immunised chimpanzeewere observed after stimulation by these three peptides, a) in thechimpanzee 60% of the lymphocytic lines obtained are of the CD8⁺phenotype,

b)in the mouse, the injection of one or other of these peptides makes itpossible immunologically to “prime” the immune system of the mouse andto obtain the production of a high level of antibodies against the Bepitope of the LSA-R peptide after the injection of the recombinantprotein LSA-R-NR. The identification of those epitopes capable ofstimulating the T lymphocytes is of great important in as much as it hasbeen established in the malaria of rodents that the protection inducedby irradiated sporozoites is dependent on the production of lymphocytescytotoxic for the infected hepatocyte, and capable of destroying them.

Furthermore, the study of 120 sera of African subjects also shows thatthe peptide LSA-NR defines a B epitope, distinct from that which isfound in the repeats, recognized by about 65% of the subjects studied.

On the other hand, the peptide LSA-TER does not constitute a significantB epitope, it is rarely recognized by the antibodies of the subjectsstudied.

The potential usefulness of the T epitopes of the LSA, in particularthose contained in the non-repetitive part, is, in addition stronglyreinforced by the results obtained in the chimpanzee:

in the chimpanzee which has been immunised by three injections atintervals of 15 days of a mixture of two recombinant proteins adsorbedon alum, the recombinant protein LSA-R-RN (clone DG536) on the one handand the recombinant protein designated DG729S (clone DG729S which formspart of the 120 clones mentioned above) on the other, it was possible toobtain several significant results:

-   -   the production of antibodies specific for each of the two        recombinant molecules, i.e. which react with the protein        LSA-R-NR and the synthetic peptides, as well as with the        recombinant protein 729S, was detected.    -   a specific proliferative response of the lymphocytes was        obtained towards the peptide LSA-NR and also to the peptides        LSA-J and LSA-TER. Sixty percent of the proliferating        lymphocytes are of the CD8⁺ phenotype, which corresponds in        particular to cytotoxic T lymphocytes.    -   After immunization of the chimpanzees, a test infection by        intravenous injection of 28 millions of sporozoites of P.        falciparum was carried out on an immunized chimpanzee and in a        control chimpanzee (which received an unrelated control        recombinant protein) and liver biopsies were made on day 6 after        infection. The examination of the biopsies showed the existence        of a cellular reaction, lympho-monocytic, around the hepatic        schizonts, infiltrating the schizonts and capable of destroying        them. Such images were not observed in the chimpanzee which had        received the control antigen.

This cellular reaction reveals the existence of T epitopes in theinjected molecules 729S and LSA-R-NR (DG536) which are capable of beingexpressed in the injected molecules, capable of being expressed by thehepatic schizonts and of inducing a cellular afflux; this result is inagreement with the results of the lymphocytic proliferation assays;finally it shows that the immune response induced by the injectedrecombinant proteins is capable, upon penetration of the parasite, ofinducing a cellular afflux, itself capable of contributing to thedefence of the organism by destroying the parasites located within thehepatocytes.

A specific proliferative response of the lymphocytes was also obtainedtowards the peptides 729S-NRI and 729S-NRII.

The cytolytic capacity of the lymphocytes of this immunized andprotected chimpanzee was measured in vitro in the following manner:lymphocyte lines were produced by in vitro stimulation with the peptidesLSA-NR and LSA-J as well as with 729-NRI and NRII in the presence ofinterleukin 2. These lines were maintained for 4 weeks by restimulationin the presence of autologous mononucleated cells from the samechimpanzee by the same peptides and interleukin 2. A cytolysis test wascarried out by incubating the irradiated peripheral mononucleated cellsof the chimpanzee with the same peptides, then after labelling withchromium 51, by incubating these cells with the lines produced. Releaseof the chromium 51 reflecting the destruction of the target cells wasobserved. These results demonstrate that the T epitopes, defined above,are capable of activating the cytolytic T lymphocytes specific for thepolypeptide sequences in question.

The antigenic and/or immunological specificities of the polypeptides ofthe invention are the following:

-   -   the polypeptide 536:        -   is recognized:        -   by antibodies originating from subjects with malaria,        -   by sera of chimpanzees immunized with the recombinant            complete protein 536,        -   co-reacts with the polypeptides described in Guérin-Marchand            et al. (Nature, cited above) by the intermediary of the            repeat sequences,        -   induces the function of antibodies (presence of B epitopes)        -   induces proliferative responses of T lymphocytes (presence            of T epitope) in the chimpanzee and in man.    -   the polypeptides NR and TER include a major T epitope and a B        epitope for man as for the immunized animal (mouse and        chimpanzee),    -   the polypeptide LSA-J includes a B epitope distinct from that of        the 17 amino acid repeat probably on account of the substitution        of S by R, and contains a T epitope recognized in man and the        chimpanzee.

The recombinant protein 729S:

-   -   is recognized:        -   by antibodies of subjects exposed to malaria,        -   by sera of chimpanzees and mice immunized by the protein            729S    -   is better recognized by the individuals capable of resisting        infection by malaria than by those who do not resist (in the        north of Senegal, the inventors have administered a radical        course of treatment of chloroquine to 100 individuals, and        followed the repositivation of the blood during the period of        transmission, from September to December a) in the subjects who        are not positive, no antibody against the protein 729S was        detected, b) in more than half of those who had not become        positive again, there existed a high titer response to the        protein 729S).    -   is recognized by the sera of three individuals who had been        vaccinated by multiple injections of sporozoites of P.        falciparum and who had resisted a test injection of        non-irradiated virulent sporozoites, but is not recognized by        the sera of four other individuals who had been vaccinated by        multiple injections of sporozoites irradiated with high doses of        radiation and who had not resisted the same test injections of        non-irradiated sporozoites. This recognition relates in        particular to the reaction of the antibodies with the        polypeptide 729S-NRII. Hence there exists a tight correlation        between the immune response directed against the molecule 729S        and the protection induced against malaria in man.

The polypeptides 729S-NRI and 729S-NRII bear additionally major Tepitopes which are recognized by the majority of these subjects whoselymphocytes were studied recently in Madagascar and Senegal (18 out of20 positives studied in Madagascar and 26 out of 46 studied in Senegal).

The polypeptide 729S-R also contains a T epitope recognized by a highproportion of individuals of Senegalese and Madagascan origin (30 to 60%of the individuals studied) but in addition it defines a major B epitopeof the molecule because the antibodies of 96 to 100% of the subjectsstudied in Senegal, Cameroon and Madagascar recognize this peptide andbecause the antibody titer observed is extremely high.

It will immediately be apparent to the specialist skilled in the artthat in the nucleotide sequences mentioned above, some of thenucleotides may be replaced by others on account of the degeneracy ofthe genetic code without the peptides being modified in any way. All ofthese nucleotide sequences as well as those which code for polypeptideswhich differ from former by one or more amino acids without theirintrinsic immunogenic activity being similarly modified, form part ofthe invention. Naturally, the same holds for the nucleotide sequenceswhich may be reconstituted and which are capable of coding for oligomerssuch as defined hereafter. The monomeric motifs are linked directly endto end or through the intermediary of peptide sequences without anyeffect on the immunogenic properties of the oligomers thus formed.

Bacteria harbouring the above-mentioned clones DG199 and DG307 weredeposited with the Collection Nationale des Cultures de Microorganismesat the Pasteur Institute in Paris (CNCM) on 22 Jul. 1986 under thenumbers I-580 and I-581, respectively. Bacteria harbouring the cloneDG145 was deposited on 15 Sep. 1986 under the number I-606. The clonesDG 536 and DG 729 were deposited on 17 Jan. 1991 under the number I-1027and on 17 Jan. 1991 under the number I-1028, respectively.

In the preceding formula, use is made of the international nomenclaturewhich designates each of the naturally occurring amino acids by a singleletter, in particular according to the table of correspondances whichfollows:

-   M Methionine-   L Leucine-   I Isoleucine-   V Valine-   F Phenylalanine-   S Serine-   P Proline-   T Threonine-   A Alanine-   Y Tyrosine-   H Histidine-   Q Glutamine-   N Asparagine-   K Lysine-   D Aspartic acid-   E Glutamic acid-   C Cysteine-   W Tryptophan-   R Arginione-   G Glycine

1. A purified polypeptide comprising at least one T epitope from aliver-stage specific protein produced by P. falciparum, wherein theamino acid sequence of said T epitope is selected from the group of theamino acid sequences of SEQ ID NOS: 41, 45 and
 46. 2. The polypeptide ofclaim 1, further comprising at least one B epitope from a liver-stagespecific protein produced by P. falciparum.
 3. A synthetic polypeptidecomprising at least one T epitope from a liver-stage specific proteinproduced by P. falciparum, wherein the amino acid sequence of said Tepitope is selected from the group of the amino acid sequences of SEQ IDNOS: 41, 45 and
 46. 4. An immunogenic composition directed againstmalaria comprising a molecule having one or more peptide sequencesbearing all or part of one or more T epitopes resulting from theinfectious activity of P. falciparum in hepatic cells, wherein said Tepitope is selected from the group of: the amino acid sequence of SEQ IDNOS: 19, 41, 42, 45 and
 46. 5. An immunogenic composition directedagainst malaria comprising a molecule having one or more peptidesequences bearing all or part of one or more T epitopes resulting fromthe infectious activity of P. falciparum in hepatic cells, wherein saidT epitope is selected from the group of: the amino acid sequence of SEQID NOS: 19, 41 and 45.